Hypoxia suppresses T-cell responses, and blockade of adenosine signaling can reverse the metabolic switch induced by hypoxia. (A) Cumulative data and representative carboxyfluorescein diacetate succinimidyl ester profiles of CD4⁺ (left panel, n = 19) and CD8⁺ (right panel, n = 19) cells after 3 days of culture in normoxia or hypoxia in the presence of anti-CD3 (0.5 µg/mL) and anti-CD28 (1 µg/mL) antibodies and the A2A agonist CGS21680 or antagonist SCH58261 (both at 1 µM, added at the beginning of culture). (B) RT-PCR studies showing expression of GLUT1 (SLC2A1) and MCT4 (SLC16A3, top panels), LDH (LDHA), and PKM2 (PKM2, bottom panels) in T lymphocytes sorted from PBMCs of 15 patients cultured for 5 days under normoxic or hypoxic conditions in the presence of anti-CD3 (2 µg/mL) and anti-CD28 (1 µg/mL) antibodies and IL-2 (100 U/mL). Where indicated, the A2A antagonist SCH58261 (10 µM) was added for the entire length of the experiment. (C) Bioenergetics profile (top panel) of dynamic measurement of the ECAR on activated T lymphocytes cultured for 5 days under normoxia or hypoxia with and without SCH58261 (10 µM). Glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM) were added as indicated by the dashed lines. Boxplots in the bottom panels show cumulative data of basal ECAR and maximal glycolysis (after glucose and oligo injection respectively) of CLL T lymphocytes (n = 4) obtained after activation and sorting as described above. Each experiment was performed in triplicate. Statistical differences were determined using the Wilcoxon signed rank and Mann-Whitney U tests followed by the Tukey test for panels A and B. B, basal; CGS, CGS21680; H, hypoxia (gray boxes); N, normoxia (open boxes); Oligo, oligomycin; SCH, SCH58261; 2-DG, 2-deoxy-d-glucose.